J Neuroimmune Pharmacol DOI 10.1007/s11481-015-9593-1
ORIGINAL ARTICLE
Endocannabinoid Catabolic Enzymes Play Differential Roles in Thermal Homeostasis in Response to Environmental or Immune Challenge Sara R. Nass & Jonathan Z. Long & Joel E. Schlosburg & Benjamin F. Cravatt & Aron H. Lichtman & Steven G. Kinsey
Received: 1 December 2014 / Accepted: 11 February 2015 # Springer Science+Business Media New York 2015
Abstract Cannabinoid receptor agonists, such as Δ9-THC, the primary active constituent of Cannabis sativa, have antipyrogenic effects in a variety of assays. Recently, attention has turned to the endogenous cannabinoid system and how endocannabinoids, including 2-arachidonoylglycerol (2-AG) and anandamide, regulate multiple homeostatic processes, including thermoregulation. Inhibiting endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH), elevates levels of 2-AG or anandamide in vivo, respectively. The purpose of this experiment was to test the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. In separate experiments, male C57BL/6J mice were administered a MAGL or FAAH inhibitor, and then challenged with the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip) or a cold (4 °C) ambient environment. Systemic LPS administration caused a significant decrease in core body temperature after 6 h, and this hypothermia persisted for at least 12 h. Similarly, cold environment induced mild hypothermia that resolved within 30 min. JZL184 exacerbated hypothermia induced by either LPS or cold challenge, both of which effects were blocked by rimonabant, but not SR144528, indicating a CB1 cannabinoid receptor mechanism of action. In contrast, the FAAH inhibitor, PF-3845, had no S. R. Nass : S. G. Kinsey (*) Department of Psychology, West Virginia University, Morgantown, WV 26506, USA e-mail: [email protected] J. Z. Long : J. E. Schlosburg : B. F. Cravatt The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA A. H. Lichtman Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA
effect on either LPS-induced or cold-induced hypothermia. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses on their own, neither MAGL nor FAAH inhibitors affect normal body temperature. However, these endocannabinoid catabolic enzymes play distinct roles in thermoregulation following hypothermic challenges. Keywords Cannabinoid . Hypothermia . Environmental stress . FAAH . MAGL . MGL Abbreviations 2-AG 2-arachidonoylglycerol AA Arachidonic acid Anandamide N-arachidonoylethanolamine CB1 Cannabinoid receptor type 1 CB2 Cannabinoid receptor type 2 FAAH Fatty acid amide hydrolase JZL184 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5yl(hydroxy)methyl)piperidine-1-carboxylate MAGL Monoacylglycerol lipase Tb Body temperature THC Δ9-tetrahydrocannabinol TNFα Tumor necrosis factor α
Introduction Thermoregulation is an essential homeostatic process that maintains core body temperature (Tb) in the presence of environmental or physiological stressors (e.g., extreme ambient temperatures) (Romanovsky 2007). A hypothermic response is elicited when thermoregulation is dysregulated (Wong 1983). A stressor, such as cold ambient temperature or systemic inflammation, triggers an inhibition of the
predominantly warm thermosensitive neurons in the preoptic anterior hypothalamus (POAH), leading to an increased response of the sympathetic nervous system (i.e., vasoconstriction, and increased oxygen consumption, respiratory rate, heart rate, and blood pressure) to promote thermogenesis and return the organism to homeostasis (Wong 1983; Romanovsky 2007). In rodents, hypothermia is induced by administration of exogenous cannabinoids such as Δ9-tetrahydrocannabinol (THC), the primary active constituent of Cannabis sativa (Freeman and Martin 1983). Cannabinoid-induced hypothermia is a well characterized phenomenon (Freeman and Martin 1983) and is a component of the Btetrad^ screening battery for cannabinoid effects (Wiley and Martin 2003). Pretreatment with a cannabinoid type 1 (i.e., CB1) receptor antagonist blocks this decrease in core body temperature, indicating that CB1 is required for cannabinoid-induced hypothermia (Wiley and Martin 2003). Indeed, CB1 selective agonists or mixed CB1/CB2 agonists induce hypothermia, whereas highly selective CB2 receptor agonists, such as O-3223, do not induce hypothermia (Kinsey et al. 2011). In addition to inducing hypothermia, cannabinoids also modulate thermoregulation in response to endotoxin challenge. For example, the non-selective cannabinoid receptor agonist WIN 55,212-2 dose-dependently attenuates fever induced by the gram negative bacterial endotoxin lipopolysaccharide (LPS), in rats. This anti-pyrogenic effect of WIN 55, 212-2 is blocked by the selective CB1 receptor antagonist rimonabant, but not the CB2 selective antagonist SR144528, indicating the necessity of CB1 in mediating the antipyrogenic effect of WIN 55,212-2 (Benamar et al. 2007). Conversely, the role of endogenous cannabinoids in thermoregulation is not well defined. Unlike exogenous cannabinoids, manipulation of the endocannabinoid system does not induce hypothermia. For example OL-135, inhibits fatty acid amide hydrolase ( FA A H ) , t h e p r i m a r y c a t a b o l i c e n z y m e f o r t h e endocannabinoid anandamide, thereby increasing brain levels of anandamide, but has no effect on body temperature (Lichtman et al. 2004). Similarly, increased brain levels of the other well characterized endocannabinoid 2arachidonoylglycerol (2-AG) occur after inhibition of its primary catabolic enzyme, monoacylglycerol lipase (MAGL) (Blankman et al. 2007). The highly selective MAGL inhibitor JZL184 increases brain 2-AG levels, but does not affect body temperature (Long et al. 2009a). When administered in a vehicle consisting of 4:1 parts polyethylene glycol (PEG300) and Polysorbate 80 (Tween80), JZL184 induced a mild reduction in Tb (Long et al. 2009b). It is noteworthy that the PEG300 vehicle, administered alone, also produced a mild hypothermic response, which was greatly augmented by JZL184 in PEG (Long et al. 2009b). However, the 1:1:18 vehicle is devoid of hypothermic
effects and JZL184 did not alter body temperature when administered in 1:1:18 vehicle (Long et al. 2009a). The following studies were designed to determine whether inhibition of MAGL or FAAH disrupts thermoregulation following physical (i.e., cold ambient temperature) or physiological (i.e., endotoxin) challenge. First, we assessed whether the MAGL inhibitor JZL184 or the FAAH inhibitor PF-3845 potentiates LPS-induced hypothermia. Second, we examined whether JZL184 or PF-3845 potentiates hypothermia induced by cold ambient temperature. Finally, we determined the contribution of CB1 and CB2 receptors in these assays.
Materials and Methods Animals Adult male C57BL/6J mice weighing approximately 25 g at the start of the experiments were singly housed and maintained on a 12:12 light cycle in a temperature (20–22 °C) and humidity controlled facility, with ad libitum access to food and water. Mice were randomly assigned to treatment groups. All experimental protocols were approved by the Institutional Animal Care and Use Committees at West Virginia University and Virginia Commonwealth University. Experimenter was blinded to drug treatment conditions. Endotoxin Challenge Baseline rectal temperature was recorded using a lubricated rectal thermocouple probe attached to a BAT12 thermometer (Thomas Scientific, Swedesboro, NJ), and after 2 h, Tb was again taken, and the mice were injected with lipopolysaccharide (LPS) dissolved in saline (2 mg/kg, ip) or saline. Tb was taken 2, 4, 6, 8, 12, and 24 h after LPS injection (Benamar et al. 2007). For the endocannabinoid studies, baseline Tb was taken, and then the mice were injected with JZL184 (1, 4, 16, 40 mg/kg, ip), PF-3845 (10 mg/kg, ip), or vehicle. These high doses JZL184 (i.e., 40 mg/kg) (Kinsey et al. 2009, 2013; Long et al. 2009b; Nomura et al. 2011) and PF-3845 (i.e., 10 mg/ kg) (Ahn et al. 2009) are sufficient to fully inhibit MAGL and FAAH, respectively, in mice. In a third study, mice were pretreated with the CB 1 receptor selective antagonist rimonabant (SR141716A, 3 mg/kg, ip) (Rinaldi-Carmona et al. 1994), the CB2 receptor selective antagonist SR144528 (3 mg/kg, ip) (Rinaldi-Carmona et al. 1998), or vehicle 30 min prior to administration of JZL184, PF-3845, or vehicle. Cold Challenge Baseline rectal temperature was recorded, and then the mice were injected (ip) with rimonabant (3 mg/kg), SR144528 (3 mg/kg), or vehicle. Thirty min later, mice were injected ip
with JZL184 (40 mg/kg), PF-3845 (10 mg/kg), or vehicle. After 2 h, core Tb was taken, and the mice were placed in a cold (5±1 °C) room for a 4 h duration. Tb was recorded every 60 min. At 4 h, the mice were removed from the cold room and returned to the 20 °C laboratory environment. Tb was taken 30, 60, and 120 min after the mice were removed from the cold room.
Drugs Rimonabant (SR141716; SR1) and SR144528 (SR2) were generously provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). The MAGL inhibitor JZL184 (Long et al. 2009b) and the FAAH inhibitor PF-3845 (Ahn et al. 2009) were synthesized in the Cravatt laboratory, as described previously. LPS from Escherichia coli 026:B6 were purchased from Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in a vehicle consisting of ethanol, Alkamuls-620 (Rhone-Poulenc, Princeton, NJ), and saline in a ratio of 1:1:18 parts, and LPS was dissolved in normal saline (i.e., 0.9 % NaCl). Doses were based on published reports (Ahn et al. 2009; Kinsey et al. 2009; Nomura et al. 2011) and administered at a volume of 10 μl/g body mass. Solutions were warmed to RT prior to injection.
Differences were considered statistically significant at p
ORIGINAL ARTICLE
Endocannabinoid Catabolic Enzymes Play Differential Roles in Thermal Homeostasis in Response to Environmental or Immune Challenge Sara R. Nass & Jonathan Z. Long & Joel E. Schlosburg & Benjamin F. Cravatt & Aron H. Lichtman & Steven G. Kinsey
Received: 1 December 2014 / Accepted: 11 February 2015 # Springer Science+Business Media New York 2015
Abstract Cannabinoid receptor agonists, such as Δ9-THC, the primary active constituent of Cannabis sativa, have antipyrogenic effects in a variety of assays. Recently, attention has turned to the endogenous cannabinoid system and how endocannabinoids, including 2-arachidonoylglycerol (2-AG) and anandamide, regulate multiple homeostatic processes, including thermoregulation. Inhibiting endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH), elevates levels of 2-AG or anandamide in vivo, respectively. The purpose of this experiment was to test the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. In separate experiments, male C57BL/6J mice were administered a MAGL or FAAH inhibitor, and then challenged with the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip) or a cold (4 °C) ambient environment. Systemic LPS administration caused a significant decrease in core body temperature after 6 h, and this hypothermia persisted for at least 12 h. Similarly, cold environment induced mild hypothermia that resolved within 30 min. JZL184 exacerbated hypothermia induced by either LPS or cold challenge, both of which effects were blocked by rimonabant, but not SR144528, indicating a CB1 cannabinoid receptor mechanism of action. In contrast, the FAAH inhibitor, PF-3845, had no S. R. Nass : S. G. Kinsey (*) Department of Psychology, West Virginia University, Morgantown, WV 26506, USA e-mail: [email protected] J. Z. Long : J. E. Schlosburg : B. F. Cravatt The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA A. H. Lichtman Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA
effect on either LPS-induced or cold-induced hypothermia. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses on their own, neither MAGL nor FAAH inhibitors affect normal body temperature. However, these endocannabinoid catabolic enzymes play distinct roles in thermoregulation following hypothermic challenges. Keywords Cannabinoid . Hypothermia . Environmental stress . FAAH . MAGL . MGL Abbreviations 2-AG 2-arachidonoylglycerol AA Arachidonic acid Anandamide N-arachidonoylethanolamine CB1 Cannabinoid receptor type 1 CB2 Cannabinoid receptor type 2 FAAH Fatty acid amide hydrolase JZL184 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5yl(hydroxy)methyl)piperidine-1-carboxylate MAGL Monoacylglycerol lipase Tb Body temperature THC Δ9-tetrahydrocannabinol TNFα Tumor necrosis factor α
Introduction Thermoregulation is an essential homeostatic process that maintains core body temperature (Tb) in the presence of environmental or physiological stressors (e.g., extreme ambient temperatures) (Romanovsky 2007). A hypothermic response is elicited when thermoregulation is dysregulated (Wong 1983). A stressor, such as cold ambient temperature or systemic inflammation, triggers an inhibition of the
predominantly warm thermosensitive neurons in the preoptic anterior hypothalamus (POAH), leading to an increased response of the sympathetic nervous system (i.e., vasoconstriction, and increased oxygen consumption, respiratory rate, heart rate, and blood pressure) to promote thermogenesis and return the organism to homeostasis (Wong 1983; Romanovsky 2007). In rodents, hypothermia is induced by administration of exogenous cannabinoids such as Δ9-tetrahydrocannabinol (THC), the primary active constituent of Cannabis sativa (Freeman and Martin 1983). Cannabinoid-induced hypothermia is a well characterized phenomenon (Freeman and Martin 1983) and is a component of the Btetrad^ screening battery for cannabinoid effects (Wiley and Martin 2003). Pretreatment with a cannabinoid type 1 (i.e., CB1) receptor antagonist blocks this decrease in core body temperature, indicating that CB1 is required for cannabinoid-induced hypothermia (Wiley and Martin 2003). Indeed, CB1 selective agonists or mixed CB1/CB2 agonists induce hypothermia, whereas highly selective CB2 receptor agonists, such as O-3223, do not induce hypothermia (Kinsey et al. 2011). In addition to inducing hypothermia, cannabinoids also modulate thermoregulation in response to endotoxin challenge. For example, the non-selective cannabinoid receptor agonist WIN 55,212-2 dose-dependently attenuates fever induced by the gram negative bacterial endotoxin lipopolysaccharide (LPS), in rats. This anti-pyrogenic effect of WIN 55, 212-2 is blocked by the selective CB1 receptor antagonist rimonabant, but not the CB2 selective antagonist SR144528, indicating the necessity of CB1 in mediating the antipyrogenic effect of WIN 55,212-2 (Benamar et al. 2007). Conversely, the role of endogenous cannabinoids in thermoregulation is not well defined. Unlike exogenous cannabinoids, manipulation of the endocannabinoid system does not induce hypothermia. For example OL-135, inhibits fatty acid amide hydrolase ( FA A H ) , t h e p r i m a r y c a t a b o l i c e n z y m e f o r t h e endocannabinoid anandamide, thereby increasing brain levels of anandamide, but has no effect on body temperature (Lichtman et al. 2004). Similarly, increased brain levels of the other well characterized endocannabinoid 2arachidonoylglycerol (2-AG) occur after inhibition of its primary catabolic enzyme, monoacylglycerol lipase (MAGL) (Blankman et al. 2007). The highly selective MAGL inhibitor JZL184 increases brain 2-AG levels, but does not affect body temperature (Long et al. 2009a). When administered in a vehicle consisting of 4:1 parts polyethylene glycol (PEG300) and Polysorbate 80 (Tween80), JZL184 induced a mild reduction in Tb (Long et al. 2009b). It is noteworthy that the PEG300 vehicle, administered alone, also produced a mild hypothermic response, which was greatly augmented by JZL184 in PEG (Long et al. 2009b). However, the 1:1:18 vehicle is devoid of hypothermic
effects and JZL184 did not alter body temperature when administered in 1:1:18 vehicle (Long et al. 2009a). The following studies were designed to determine whether inhibition of MAGL or FAAH disrupts thermoregulation following physical (i.e., cold ambient temperature) or physiological (i.e., endotoxin) challenge. First, we assessed whether the MAGL inhibitor JZL184 or the FAAH inhibitor PF-3845 potentiates LPS-induced hypothermia. Second, we examined whether JZL184 or PF-3845 potentiates hypothermia induced by cold ambient temperature. Finally, we determined the contribution of CB1 and CB2 receptors in these assays.
Materials and Methods Animals Adult male C57BL/6J mice weighing approximately 25 g at the start of the experiments were singly housed and maintained on a 12:12 light cycle in a temperature (20–22 °C) and humidity controlled facility, with ad libitum access to food and water. Mice were randomly assigned to treatment groups. All experimental protocols were approved by the Institutional Animal Care and Use Committees at West Virginia University and Virginia Commonwealth University. Experimenter was blinded to drug treatment conditions. Endotoxin Challenge Baseline rectal temperature was recorded using a lubricated rectal thermocouple probe attached to a BAT12 thermometer (Thomas Scientific, Swedesboro, NJ), and after 2 h, Tb was again taken, and the mice were injected with lipopolysaccharide (LPS) dissolved in saline (2 mg/kg, ip) or saline. Tb was taken 2, 4, 6, 8, 12, and 24 h after LPS injection (Benamar et al. 2007). For the endocannabinoid studies, baseline Tb was taken, and then the mice were injected with JZL184 (1, 4, 16, 40 mg/kg, ip), PF-3845 (10 mg/kg, ip), or vehicle. These high doses JZL184 (i.e., 40 mg/kg) (Kinsey et al. 2009, 2013; Long et al. 2009b; Nomura et al. 2011) and PF-3845 (i.e., 10 mg/ kg) (Ahn et al. 2009) are sufficient to fully inhibit MAGL and FAAH, respectively, in mice. In a third study, mice were pretreated with the CB 1 receptor selective antagonist rimonabant (SR141716A, 3 mg/kg, ip) (Rinaldi-Carmona et al. 1994), the CB2 receptor selective antagonist SR144528 (3 mg/kg, ip) (Rinaldi-Carmona et al. 1998), or vehicle 30 min prior to administration of JZL184, PF-3845, or vehicle. Cold Challenge Baseline rectal temperature was recorded, and then the mice were injected (ip) with rimonabant (3 mg/kg), SR144528 (3 mg/kg), or vehicle. Thirty min later, mice were injected ip
with JZL184 (40 mg/kg), PF-3845 (10 mg/kg), or vehicle. After 2 h, core Tb was taken, and the mice were placed in a cold (5±1 °C) room for a 4 h duration. Tb was recorded every 60 min. At 4 h, the mice were removed from the cold room and returned to the 20 °C laboratory environment. Tb was taken 30, 60, and 120 min after the mice were removed from the cold room.
Drugs Rimonabant (SR141716; SR1) and SR144528 (SR2) were generously provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). The MAGL inhibitor JZL184 (Long et al. 2009b) and the FAAH inhibitor PF-3845 (Ahn et al. 2009) were synthesized in the Cravatt laboratory, as described previously. LPS from Escherichia coli 026:B6 were purchased from Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in a vehicle consisting of ethanol, Alkamuls-620 (Rhone-Poulenc, Princeton, NJ), and saline in a ratio of 1:1:18 parts, and LPS was dissolved in normal saline (i.e., 0.9 % NaCl). Doses were based on published reports (Ahn et al. 2009; Kinsey et al. 2009; Nomura et al. 2011) and administered at a volume of 10 μl/g body mass. Solutions were warmed to RT prior to injection.
Differences were considered statistically significant at p
